Determination method for enzyme activity of DNA ligase and related compound

Abstract

The invention particularly relates to a determination method of DNA ligase and a related compound. The determination method comprises the following steps that a single-chain sequence MB is provided, at least the 5'end and the 3 'end of the single-chain sequence MB are DNA sequences and are complementarily paired, a neck ring structure can be formed, and the 5' end and the 3 'end of the single-chain sequence MB are provided with a fluorophore and a quenching group respectively; providing two single-stranded DNA sequences D1 and D2, wherein the two single-stranded DNA sequences D1 and D2 are complementary and adjacent to the annular part of the single-stranded sequence MB; and hybridizing the two single-stranded DNA sequences D1 and D2 with the single-stranded sequence MB to form a compound with a neck ring. Respectively treating the compound by using a DNA ligase standard substance to obtain a standard curve, and substituting the detection result of the DNA ligase to be detected into the standard curve. The method is simple to operate and does not have a fluorescence background value, the activity of the ligase can be judged by directly depending on the intensity of the generated fluorescence, and the comparison is more convenient and more sensitive.

Description

Technical Field

[0001] The present invention relates to the technical field of biochemical detection, and in particular to a method for determining the activity of a DNA ligase and a complex involved therein. Background Art

[0002] The most commonly used method for measuring DNA ligase activity is capillary electrophoresis, which analyzes the amount of unligated and ligated fragments in the ligation reaction product to determine ligase activity. This method is relatively complex, costly, and requires specialized instrumentation. Therefore, the development of new DNA ligase activity assays is of great significance.

[0003] The invention patent with publication number CN 113061645A discloses a method for determining the activity of a DNA ligase, comprising: (1) providing an RNA single strand and a complementary acceptor DNA single strand and a donor DNA single strand; (2) annealing to obtain a DNA / RNA hybrid double strand, wherein the 3'-terminal hydroxyl group of the acceptor DNA single strand is adjacent to the 5'-terminal phosphate group of the donor DNA single strand but has a nick, and the fluorescent group of the hybrid double strand produces fluorescence; (3) treating the DNA / RNA hybrid double strand with a DNA ligase to be tested to obtain a complete DNA / RNA hybrid double strand connected at the nick, and the fluorescent group of the hybrid double strand maintains fluorescence; (4) directly or heat-denaturing the complete DNA / RNA hybrid double strand and then digesting it with ribonuclease to leave a DNA ligation product, and quantifying the fluorescence intensity of the product to determine the activity of the DNA ligase to be tested.

[0004] This method is similar to some existing detection methods. It not only requires the addition of RNase H to digest the RNA in the DNA / RNA hybrid chain, or heat denaturation followed by RNase digestion of the RNA, ultimately leaving behind a DNA ligation product, which is a relatively complex operation, but also uses the intensity of fluorescence quenching to determine the ligation efficiency. Due to the presence of fluorescence background value, the method of relying on the intensity of fluorescence quenching to determine is not simple and sensitive enough. Summary of the Invention

[0005] Based on this, the present invention provides a new method for determining the enzyme activity of DNA ligase. The method is simple to operate and does not have a fluorescence background value. The activity of the ligase can be directly judged based on the magnitude of the generated fluorescence intensity, which is more convenient and more sensitive.

[0006] The present invention adopts the following technical solution to achieve the above technical purpose: a method for determining the activity of DNA ligase, comprising the following steps:

[0007] (1) Preparing a complex: providing a single-stranded sequence MB, wherein at least the 5' end and the 3' end of the single-stranded sequence MB are DNA sequences and are complementary to each other to form a neck loop structure, and the 5' end and the 3' end of the single-stranded sequence MB have a fluorescent group and a quenching group, respectively; providing two single-stranded DNA sequences D1 and D2, wherein the two single-stranded DNA sequences D1 and D2 are complementary to and adjacent to the loop portion of the single-stranded sequence MB; hybridizing the two single-stranded DNA sequences D1 and D2 with the single-stranded sequence MB to form a complex with a neck loop, thereby obtaining;

[0008] (2) The standard is diluted in a gradient manner, and the fluorescence value f1 after the ligation reaction of different amounts of ligase standard is detected by a microplate reader. The fluorescence value f0 of the no-enzyme control is then detected, and the net fluorescence value f = f1-f0 is calculated. A standard curve is calculated between the amount of standard m and the net fluorescence value f;

[0009] (3) The enzyme to be tested is diluted in a gradient manner, and the fluorescence value F1 after the ligation reaction is detected by a microplate reader with different input amounts of the ligase to be tested, and the fluorescence value F0 of the no-enzyme control is detected, and the net fluorescence value F=F1-F0 is calculated;

[0010] (4) Substitute the net fluorescence value C corresponding to each gradient in the linear range of the tested ligase into the standard curve of the standard sample to calculate the theoretically estimated enzyme activity value. Divide this enzyme activity value by the amount of the tested enzyme input in each gradient to obtain the corresponding specific activity. Calculate the mean of the gradient specific activity of the tested enzyme and convert it to U / μL to obtain the final enzyme specific activity.

[0011] As a preferred embodiment, in step (2), the standard DNA ligase is diluted 2-fold using enzyme dilution buffer to obtain 7 dilutions of different concentrations, and then the ligation reaction is carried out. The net fluorescence mean obtained at the same time point with different input amounts of DNA ligase is recorded, and the standard curve is fitted.

[0012] As a preferred embodiment, in step (3), the DNA ligase to be tested is diluted with enzyme dilution buffer to obtain dilutions of different concentrations, and then a ligation reaction is performed. The net fluorescence mean obtained at the same time point with different input amounts of DNA ligase is recorded, and the specific activity of the DNA ligase to be tested is calculated based on the standard curve of step (2).

[0013] As a preferred embodiment, during detection in steps (2) and (3), the concentration of the complex is 3.33 μmol / L.

[0014] As a preferred embodiment, the 6 bases at the 5' end and the 6 bases at the 3' end of the single-stranded sequence MB are DNA sequences and are complementary.

[0015] As a preferred embodiment, the neck loop structure portion of the single-stranded sequence MB is a DNA sequence or an RNA sequence.

[0016] As a preferred embodiment, the fluorescent group is FAM, and the quenching group is DABCYL.

[0017] As a preferred embodiment, when used for comparison or detection of the enzymatic activities of E. coli DNA ligase, T3 DNA ligase, T4 DNA ligase, and T7 DNA ligase, the single-stranded sequence MB is: 5'-GCATGTTAGTCTCACAACTGCTGAGTCGTACA-3' (SEQ ID NO. 1), the single-stranded DNA sequence D1 is 5'-CTCAGCAGT-3', and the single-stranded DNA sequence D2 is 5'-PHO-TGTGAGACT-3';

[0018] When used for comparison or detection of PBCV DNA ligase activity, the single-stranded MB sequence is: 5'-GCATGT / rG / / rU / / rA / / rG / / rC / / rA / / rC / / rU / / rC / / rA / / rU / / rC / / rG / / rA / / rU / / rG / / rC / / rU / / rA / / rG / CGTACA-3' (SEQ ID NO. 2), the single-stranded DNA sequence D1 is: 5'-TAGCAT CGA-3', and the single-stranded DNA sequence D2 is 5'-PHO-TGAGTGCTA-3'.

[0019] The present invention also provides a complex, which is the complex involved in the above method.

[0020] The present invention also provides the use of the above-mentioned complex in the comparison or detection of DNA ligase activity, wherein, in the complex, the fluorescent group and the quenching group are adjacent to each other and do not generate fluorescence. Under the action of the ligase to be tested, the single-stranded DNA sequences D1 and D2 undergo a ligation reaction to form a phosphodiester bond, the neck ring structure is destroyed, the fluorescent group and the quenching group are separated, and fluorescence is generated. Within a certain range, the enzyme activity is proportional to the intensity of the generated fluorescence.

[0021] Compared with the prior art, the present invention has the following beneficial effects: the present invention adopts a specific complex as a substrate. In the complex substrate, under the action of the neck ring structure, the fluorescent group and the quenching group are close to each other and no fluorescence is generated. Under the action of the ligase, the neck ring structure is destroyed, the fluorescent group and the quenching group are separated, and fluorescence is generated. Further, by deducing the linear relationship between the amount of ligation product generated and the amount of ligase input, the enzyme activity of the corresponding ligase is calculated. This method has accurate measurement, good reproducibility and no fluorescence background value. It can be directly used for the comparison of multiple enzyme activities and the determination of enzyme activity, without the need for digestion and other processing steps, and is simple to operate. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is the standard curve of the two standards of E. coli DNA ligase in Example 1;

[0023] Figure 2 is the linear range of the E. coli DNA ligase to be tested in Example 1;

[0024] Figure 3 is the standard curve of the two T3 DNA ligase standards in Example 1;

[0025] Figure 4 is the linear range of the T3 DNA ligase to be tested in Example 1;

[0026] Figure 5 is the standard curve of the two PBCV DNA ligase standards in Example 2;

[0027] Figure 6 This is the linear range of the PBCV DNA ligase to be tested in Example 2. DETAILED DESCRIPTION

[0028] The present invention provides a method for determining DNA ligase activity, comprising the following steps:

[0029] (1) Preparing a complex: providing a single-stranded sequence MB, wherein at least the 5' end and the 3' end of the single-stranded sequence MB are DNA sequences and are complementary to each other to form a neck loop structure, and the 5' end and the 3' end of the single-stranded sequence MB have a fluorescent group and a quenching group, respectively; providing two single-stranded DNA sequences D1 and D2, wherein the two single-stranded DNA sequences D1 and D2 are complementary to and adjacent to the loop portion of the single-stranded sequence MB; hybridizing the two single-stranded DNA sequences D1 and D2 with the single-stranded sequence MB to form a complex with a neck loop, thereby obtaining;

[0030] (2) The standard is diluted in a gradient manner, and the fluorescence value f1 after the ligation reaction of different amounts of ligase standard is detected by a microplate reader. The fluorescence value f0 of the no-enzyme control is then detected, and the net fluorescence value f = f1-f0 is calculated. A standard curve is calculated between the amount of standard m and the net fluorescence value f;

[0031] (3) The enzyme to be tested is diluted in a gradient manner, and the fluorescence value F1 after the ligation reaction is detected by a microplate reader with different input amounts of the ligase to be tested, and the fluorescence value F0 of the no-enzyme control is detected, and the net fluorescence value F=F1-F0 is calculated;

[0032] (4) Substitute the net fluorescence value C corresponding to each gradient in the linear range of the tested ligase into the standard curve of the standard sample to calculate the theoretically estimated enzyme activity value. Divide this enzyme activity value by the amount of the tested enzyme input in each gradient to obtain the corresponding specific activity. Calculate the mean of the gradient specific activity of the tested enzyme and convert it to U / μL to obtain the final enzyme specific activity.

[0033] In the complex in step (1) of the present invention, the fluorescent group and the quenching group are adjacent and do not generate fluorescence. Under the action of the ligase to be tested, the single-stranded DNA sequences D1 and D2 undergo a ligation reaction to form a phosphodiester bond, the neck ring structure is destroyed, the fluorescent group and the quenching group are separated, and fluorescence is generated. Within a certain range, the enzyme activity is proportional to the intensity of the generated fluorescence, thereby realizing the detection of the ligase.

[0034] In the present invention, the neck loop structure portion of the single-stranded sequence MB can be either a DNA sequence or an RNA sequence. When detected in steps (2) and (3), the concentration of the complex is preferably 3.33 μmol / L.

[0035] The present invention will be described in further detail below in conjunction with specific embodiments so that those skilled in the art can understand the present invention more clearly. The following embodiments are only used to illustrate the present invention, but are not limited to limiting the scope of the present invention. Based on the specific embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work belong to the protection scope of the present invention.

[0036] In the examples of the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art; in the examples of the present invention, unless otherwise specified, the technical means used are conventional means well known to those skilled in the art. In the examples of the present invention, the raw materials used are all conventional commercial products.

[0037] Example 1

[0038] The present invention provides a method for determining DNA ligase activity, which is as follows:

[0039] (1) Preparation of ligation reaction substrate

[0040] a. DNA single-strand MB, D1, and D2 were diluted to 10 pmol / μL with Tris HCl+NaCl solution;

[0041] Among them, the sequence of MB is: 5'-FAM-GCATGTTAGTCTCACAACTGCTGAGT CGTACA-DABCYL-3'; the sequence of D1 is: 5'-CTCAGCAGT-3'; and the sequence of D2 is: 5'-PHO-TGTGAGACT-3'.

[0042] Tris HCl+NaCl solution: 10mM Tris, pH 7.5-8.0, 50mM NaCl, 1mM EDTA.

[0043] b. Mix equal volumes of the diluted MB, D1, and D2, incubate at 95°C for 5 min in a PCR instrument, gradually cool to room temperature (-1°C / cycle, 1 min / cycle, 70 cycles), heat the lid at 105°C, and the final product concentration is 3.33 mol / L.

[0044] c. Aliquot 300 μL of the above substrate into tubes, store in a -20°C refrigerator, and thaw on ice before use.

[0045] (2) Preparation of ligation reaction solution

[0046] a. Dilute the standard and the DNA ligase to be tested by 2-fold using DNA ligase dilution solution (10 mM Tris-HCl, 300 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 50% glycerol, pH 7.4 at 25°C) to obtain seven different dilutions.

[0047] b. The preparation of ligase reaction system is shown in Table 1 below;

[0048] Table 1

[0049]

[0050]

[0051] (3) Reaction procedure

[0052] PCR instrument: 16℃ for 30 min, 65℃ for 20 min, hot cover temperature 75℃, volume 20μL.

[0053] (4) Detection of fluorescence intensity by enzyme-labeled instrument

[0054] Add 190 μL of water to the ligation reaction product, shake and mix, take 200 μL and transfer it to the ELISA plate, let it stand at room temperature for 120 minutes, and detect the fluorescence intensity with a microplate reader.

[0055] (5) Data processing

[0056] a. Plotting the standard curve: Calculate the average of two replicate wells of the standard. Subtract the average fluorescence intensity of the no-enzyme control (f0) from the average fluorescence intensity of each sample (f1) to calculate the net fluorescence mean (f). With the amount of standard ligase (m, unit: U) as the abscissa and the net fluorescence value as the ordinate, calculate the standard curve fitting equation between the standard amount m and the net fluorescence value f: f = km + b (Equation 1);

[0057] b. Data Processing for Test Enzyme Samples: Calculate the average fluorescence intensity of two replicate wells of the test enzyme sample. Subtract the average fluorescence intensity of the no-enzyme control (F0) from the average fluorescence intensity of each sample (F1) to calculate the net mean fluorescence (F). Create a scatter plot with the amount of DNA ligase input (M, ng) as the abscissa and the average fluorescence value (F) of each gradient as the ordinate. Fit a first-order linear curve to obtain a curve showing the relationship between net mean fluorescence and DNA ligase input mass, with an R² ≥ 0.99.

[0058] c. Calculation of specific activity of the enzyme to be tested

[0059] Substitute the net fluorescence mean F corresponding to the points in the linear interval of the test enzyme into the linear fitting equation of the standard curve to obtain the test DNA ligase activity (unit: U) = (Fb) / k, the test DNA ligase specific activity (unit: U / ng) = test DNA ligase activity / M = (Fb) / k / M (F: net fluorescence mean of the test enzyme sample; M: amount of the test enzyme sample; k, b: constants in Equation 1).

[0060] Note: The maximum and minimum values ​​of the fluorescence mean in the standard curve are within the reliable detection range. The fluorescence mean obtained by the DNA ligase assay should be within the detection range. Data outside the detection range should be discarded.

[0061] d. The average value of the enzyme activity calculated from each gradient of enzyme input within each repeated experiment is used as the standard value of the enzyme activity. A coefficient of variation within each repeated experiment of ≤10% is considered acceptable. Coefficient of variation = STDEV value / average value.

[0062] e. Final result: The average value obtained from three rounds of repeated experiments is the final result.

[0063] (6) Result example

[0064] A. E. coli DNA ligase activity test. The original data are shown in Table 2. The standard curve and linear fitting equation of the standard (NEB, catalog number: M0205S) are shown in Figure 1 、 2 shown.

[0065] Table 2

[0066]

[0067] The specific activity was calculated based on the standard curve of the above standards and the results of the tested E. coli DNA ligase, and the results are shown in Table 3 below.

[0068] Table 3

[0069]

[0070]

[0071] From the above results, it can be seen that within a certain range, as the input amount of E. coli DNA ligase increases, the fluorescence intensity detected by the microplate reader also shows an upward trend.

[0072] Standard 1: The linear range exists within the enzyme input range of 0.25U. The standard curve 1 is: y = 44283x + 166.45, R2 = 0.9966 ≥ 0.99;

[0073] Standard 2: There is a linear range within the enzyme input of 0.25 U. Standard curve 2 is: y = 46413x + 308.11, R2 = 0.9965 ≥ 0.99;

[0074] Enzyme to be tested: The linear range of the input amount between 5ng-0.3125ng is y=2058.1x+531.21, R 2 =0.9937≥0.99, the measured enzyme activities were 80.59 U / μL (standard curve 1) and 72.21 U / μL (standard curve 2), with a coefficient of variation of 6.18%≤10%; the average was 75.90 U / μL.

[0075] B. T3 DNA ligase activity test. The raw data are shown in Table 4. The standard curve and linear fitting equation of the standard (NEB, catalog number: M0317S) are shown in Table 4. Figure 3 、 4 shown.

[0076] Table 4

[0077]

[0078]

[0079] The specific activity was calculated based on the standard curve of the above standards and the results of the tested E. coli DNA ligase, and the results are shown in Table 5 below.

[0080] Table 5

[0081]

[0082] From the above results, it can be seen that within a certain range, as the amount of T3 DNA ligase input increases, the fluorescence intensity detected by the microplate reader also shows an upward trend.

[0083] Standard 1: The linear range exists within the enzyme input range of 1.5 U. The standard curve 1 is: y = 10739x + 151.78, R2 = 0.997 ≥ 0.99;

[0084] Standard 2: When the enzyme dosage is less than 1.5 U, the linear range exists. Standard curve 2 is: y = 12295x + 109.36, R2 = 0.999 ≥ 0.99;

[0085] Enzyme to be tested: There is a linear range y=40009x-189.87 between the input amount of 0.25ng-0.015625ng, R2=0.999≥0.99, the measured enzyme activity is 14500.54U / μL (standard curve 1) and 13093.24U / μL (standard curve 2), the coefficient of variation is 5.10%≤10%; the average is 13796.89U / μL.

[0086] Example 2

[0087] This embodiment provides a method for determining the activity of PBCV DNA ligase, comprising the following steps:

[0088] (1) Preparation of ligation reaction substrate

[0089] a. Single-chain MB, D1, and D2 were diluted to 10 pmol / L with TrisHCl+NaCl solution;

[0090] The sequence of the single-chain MB is: 5'-GCATGT / rG / / rU / / rA / / rG / / rC / / rA / / rC / / rU / / rC / / rA / / rU / / rC / / rG / / rA / / rU / / rG / / rC / / rU / / rA / / rG / CGTACA-3',

[0091] Sequence D1 is: 5'-TAGCATCGA-3',

[0092] The sequence D2 is 5'-PHO-TGAGTGCTA-3'.

[0093] The preparation method of TrisHCl+NaCl solution is: 10mM Tris, pH 7.5-8.0, 50mM NaCl, 1mM EDTA.

[0094] b. Mix equal volumes of the diluted MB, D1, and D2, incubate at 95°C for 5 min in a PCR instrument, gradually cool to room temperature (-1°C / cycle, 1 min / cycle, 10 cycles), heat the lid at 105°C, and the final product concentration is 3.33 pmol / L.

[0095] c. Aliquot 300 μL of the above substrate into tubes and store at -20°C. Thaw on ice before use. Note: Experiments should be performed at 4°C.

[0096] (2) Preparation of ligation reaction solution

[0097] a. Dilute the standard (NEB, Cat. No. M0375S) and the PBCV DNA ligase to be tested by 2-fold dilution using DNA ligase dilution solution (10 mM Tris-HCl, 300 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 50% glycerol, pH 7.4 @ 25°C) to obtain seven different dilutions.

[0098] b. The preparation of ligase reaction system is shown in Table 6:

[0099] Table 6

[0100]

[0101]

[0102] (3) Reaction procedure

[0103] PCR instrument: 16℃ for 30 min, 65℃ for 20 min, hot cover temperature 75℃, volume 20μL.

[0104] (4) Detection of fluorescence intensity by enzyme-labeled instrument

[0105] 190 μL of water was added to the ligation reaction product, and the mixture was shaken and mixed. 200 μL of the product was transferred to an ELISA plate and the fluorescence intensity was detected by an ELISA reader.

[0106] (5) Data processing

[0107] a. Plotting the standard curve: Calculate the average of two replicate wells of the standard. Subtract the average fluorescence intensity of the no-enzyme control (f0) from the average fluorescence intensity of each sample (f1) to calculate the net fluorescence mean (f). With the amount of standard ligase (m, unit: U) as the abscissa and the net fluorescence value as the ordinate, calculate the standard curve fitting equation between the standard amount m and the net fluorescence value f: f = km + b (Equation 1);

[0108] b. Data Processing for Test Enzyme Samples: Calculate the average fluorescence intensity of two replicate wells of the test enzyme sample. Subtract the average fluorescence intensity of the no-enzyme control (F0) from the average fluorescence intensity of each sample (F1) to calculate the net mean fluorescence (F). Create a scatter plot with the amount of DNA ligase input (M, ng) as the abscissa and the average fluorescence value (F) of each gradient as the ordinate. Fit a first-order linear curve to obtain a curve showing the relationship between net mean fluorescence and DNA ligase input mass, with an R² ≥ 0.99.

[0109] c. Calculation of specific activity of the enzyme to be tested

[0110] Substitute the net fluorescence mean F corresponding to the points in the linear interval of the test enzyme into the linear fitting equation of the standard curve to obtain the test DNA ligase activity (unit: U) = (Fb) / k, the test DNA ligase specific activity (unit: U / ng) = test DNA ligase activity / M = (Fb) / k / M (F: net fluorescence mean of the test enzyme sample; M: amount of the test enzyme sample; k, b: constants in Equation 1).

[0111] Note: The maximum and minimum values ​​of the fluorescence mean in the standard curve are within the reliable detection range. The fluorescence mean obtained by the DNA ligase assay should be within the detection range. Data outside the detection range should be discarded.

[0112] d. The average value of the enzyme activity calculated from each gradient of enzyme input within each repeated experiment is used as the standard value of the enzyme activity. A coefficient of variation within each repeated experiment of ≤10% is considered acceptable. Coefficient of variation = STDEV value / average value.

[0113] e. Final result: The average value obtained from three rounds of repeated experiments is the final result.

[0114] (6) Result example

[0115] The original data are shown in Table 7 below. The standard curve and linear fitting equation of the standard are shown in Figure 5 、 6 shown.

[0116] Table 7

[0117]

[0118] The specific activity was calculated based on the standard curve of the above standards and the results of the tested PBCV DNA ligase, and the results are shown in Table 8 below.

[0119] Table 8

[0120]

[0121]

[0122] From the above results, it can be seen that within a certain range, as the amount of PBCV DNA Ligase input increases, the fluorescence intensity detected by the microplate reader also shows an upward trend.

[0123] Standard 1: When the enzyme dosage is less than 10 U, the linear range exists. Standard curve 1 is: y = 257.87x + 201.87, R2 = 0.9874 ≥ 0.985;

[0124] Standard 2: When the enzyme dosage is less than 10 U, the linear range exists. Standard curve 2 is: y = 205.04x + 419.56, R2 = 0.9866 ≥ 0.985;

[0125] Enzyme to be tested: There is a linear range y=32.324x+450.25R2=0.988≥0.985 between the input amount of 50ng-6.25ng, and the measured enzyme activity is 390.52U / μL (standard curve 1) and 331.89U / μL (standard curve 2), with a coefficient of variation of 8.12%≤10%; the average is 361.21U / μL.

[0126] It is important to note that the above embodiments are intended only to further illustrate and describe the technical solutions of the present invention and are not intended to further limit the technical solutions of the present invention. The methods of the present invention are merely preferred implementations and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A method for determining DNA ligase activity, characterized in that: The following steps are involved: (1) Preparing a complex: providing a single-stranded sequence MB, wherein at least the 5' end and the 3' end of the single-stranded sequence MB are DNA sequences and are complementary to each other to form a neck loop structure, and the 5' end and the 3' end of the single-stranded sequence MB have a fluorescent group and a quenching group, respectively; providing two single-stranded DNA sequences D1 and D2, wherein the two single-stranded DNA sequences D1 and D2 are complementary to and adjacent to the loop portion of the single-stranded sequence MB; hybridizing the two single-stranded DNA sequences D1 and D2 with the single-stranded sequence MB to form a complex with a neck loop, thereby obtaining; (2) The standard is diluted in a gradient manner, and the fluorescence value f1 after the ligation reaction of different amounts of ligase standard is detected by a microplate reader. The fluorescence value f0 of the no-enzyme control is then detected, and the net fluorescence value f = f1-f0 is calculated. A standard curve is calculated between the amount of standard m and the net fluorescence value f; (3) The enzyme to be tested is diluted in a gradient manner, and the fluorescence value F1 after the ligation reaction is detected by a microplate reader with different input amounts of the ligase to be tested, and the fluorescence value F0 of the no-enzyme control is detected, and the net fluorescence value F=F1-F0 is calculated; (4) Substitute the net fluorescence value corresponding to each gradient in the linear range of the tested ligase into the standard curve of the standard sample to calculate the theoretical enzyme activity value. Divide this enzyme activity value by the amount of the tested enzyme input in each gradient to obtain the corresponding specific activity. Calculate the mean of the gradient specific activity of the tested enzyme and convert it to U / μL to obtain the final enzyme specific activity.

2. The method according to claim 1, characterized in that In step (2), the standard DNA ligase was diluted 2-fold using enzyme dilution buffer to obtain 7 dilutions of different concentrations, and then the ligation reaction was carried out. The net fluorescence mean obtained at the same time point with different input amounts of DNA ligase was recorded, and the standard curve was fitted.

3. The method according to claim 1, characterized in that In step (3), the DNA ligase to be tested is diluted with enzyme dilution buffer to obtain dilutions of different concentrations, and then a ligation reaction is performed. The net fluorescence mean obtained at the same time point with different input amounts of DNA ligase is recorded, and the specific activity of the DNA ligase to be tested is calculated based on the standard curve of step (2).

4. The method according to claim 1, wherein During the detection in steps (2) and (3), the concentration of the complex was 3.33 μmol / L.

5. The method according to claim 1, wherein The 6 bases at the 5' end and the 6 bases at the 3' end of the single-stranded sequence MB are DNA sequences and are complementary.

6. The method according to claim 1, characterized in that The neck loop structure portion of the single-stranded sequence MB is a DNA sequence or an RNA sequence.

7. The method according to claim 1, characterized in that The fluorescent group is FAM, and the quenching group is DABCYL.

8. The method according to claim 1, characterized in that When used for comparison or detection of the enzymatic activities of E. coli DNA ligase, T3 DNA ligase, T4 DNA ligase, and T7 DNA ligase, the single-stranded sequence MB is: 5'-GCATGTTAGTCTCACAACTGCTGAGTCGTACA-3', the single-stranded DNA sequence D1 is 5'-CTCAGCAGT-3', and the single-stranded DNA sequence D2 is 5'-PHO-TGTGAGACT-3'; When used for comparison or detection of PBCV DNA ligase activity, the single-stranded MB sequence is: 5'-GCATGT / rG / / rU / / rA / / rG / / rC / / rA / / rC / / rU / / rC / / rA / / rU / / rC / / rG / / rA / / rU / / rG / / rC / / rU / / rA / / rG / CGTACA-3', the single-stranded DNA sequence D1 is: 5'-TAGCATCGA-3', and the single-stranded DNA sequence D2 is 5'-PHO-TGAGTGCTA-3'.

9. A composite, characterized in that The complex is the complex according to any one of claims 1 to 7.

10. Use of the complex according to claim 9 in comparison or detection of DNA ligase activity, wherein: Under the action of the ligase to be tested, the single-stranded DNA sequences D1 and D2 undergo a ligation reaction, the neck ring structure is destroyed, the fluorescent group and the quenching group are separated, and fluorescence is generated. Within a certain range, the enzyme activity is proportional to the intensity of the fluorescence generated.

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